Zvanysse Week 10 - Revision history

Zvanysse: Updating acknowledgments and References

2017-11-05T23:59:10Z

Updating acknowledgments and References

Zvanysse: /* Electronic Workbook */

2017-11-05T23:53:42Z

‎Electronic Workbook

Zvanysse: updating definitions

2017-11-03T21:15:00Z

updating definitions

Zvanysse: working on electronic workbook

2017-11-03T20:47:09Z

working on electronic workbook

Zvanysse: uploading zip and instructions

2017-11-03T20:40:07Z

uploading zip and instructions

Zvanysse: uploading powerpoint

2017-11-03T20:24:50Z

uploading powerpoint

New page

==Powerpoint==
[[Media:StemGraphResults.pptx|dGLN3 Stem Graph Result Powerpoint]]

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 # '''Prepare your microarray data file for loading into STEM.'''
 #* Download your Excel workbook that you used for your [[Week 8]] assignment.
-#* Insert a new worksheet into your Excel workbook, and name it "(STRAIN)_stem".
+#* Insert a new worksheet into your Excel workbook, and name it "dGLN3_stem".
 #* Select all of the data from your "dGLN3_ANOVA" worksheet and Paste special > paste values into your "dGLN3_stem" worksheet.
 #** Your leftmost column should have the column header "Master_Index".  Rename this column to "SPOT".  Column B should be named "ID".  Rename this column to "Gene Symbol".  Delete the column named "Standard_Name".
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 *#substrate-specific transporter activity: Enables the directed movement of a specific substance or group of related substances (such as macromolecules, small molecules, ions) into, out of or within a cell, or between cells.
 *#meiotic cell cycle process: Progression through the phases of the meiotic cell cycle, in which canonically a cell replicates to produce four offspring with half the chromosomal content of the progenitor cell via two nuclear divisions.

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 *#chromosome organization: A process that is carried out at the cellular level that results in the assembly, arrangement of constituent parts, or disassembly of chromosomes, structures composed of a very long molecule of DNA and associated proteins that carries hereditary information. This term covers covalent modifications at the molecular level as well as spatial relationships among the major components of a chromosome.
 *#transmembrane transport: The process in which a solute is transported across a lipid bilayer, from one side of a membrane to the other.
-*#organelle fission
+*#organelle fission: The creation of two or more organelles by division of one organelle.
-*#macromolecule modification
+*#macromolecule modification: The covalent alteration of one or more monomeric units in a polypeptide, polynucleotide, polysaccharide, or other biological macromolecule, resulting in a change in its properties.
-*#substrate-specific transporter activity
+*#substrate-specific transporter activity: Enables the directed movement of a specific substance or group of related substances (such as macromolecules, small molecules, ions) into, out of or within a cell, or between cells.
-*#meiotic cell cycle process
+*#meiotic cell cycle process: Progression through the phases of the meiotic cell cycle, in which canonically a cell replicates to produce four offspring with half the chromosomal content of the progenitor cell via two nuclear divisions.

@@ Line 54: / Line 54: @@
 ==Powerpoint and Zip==
-[[Media:StemGraphResults.pptx|dGLN3 Stem Graph Result Powerpoint]]
+[[Media:StemGraphResults.pptx|dGLN3 Stem Graph Result Powerpoint]] <br>
 [[Media:DGLN3_STEM.zip|DGLN3 Zip File]]
 ==Electronic Workbook==
 *Number of Genes left <0.05 = 1258
-*I selected '''Profile 48''' as my desired profile because it showed the clearest pattern at different time points. 55.0 genes belong to this profile and 22.0 genes were expected to belong to profile 48. The p-value for the enrichment of genes in this profile is 2.2 E-9.
+*I selected '''Profile 48''' as my desired profile because it showed the clearest pattern at different time points. 55.0 genes belong to this profile and 22.0 genes were expected to belong to profile 48. The p-value for the enrichment of genes in this profile is 2.2 E-9. There are 12 GO terms associated with this profile at p < 0.05 and 0 GO terms associated with this profile with a corrected p value < 0.05.

← Older revision		Revision as of 20:47, 3 November 2017
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	[[Media:StemGraphResults.pptx\|dGLN3 Stem Graph Result Powerpoint]]		[[Media:StemGraphResults.pptx\|dGLN3 Stem Graph Result Powerpoint]]
	[[Media:DGLN3_STEM.zip\|DGLN3 Zip File]]		[[Media:DGLN3_STEM.zip\|DGLN3 Zip File]]
		+
		+	==Electronic Workbook==
		+	*Number of Genes left <0.05 = 1258
		+	*I selected '''Profile 48''' as my desired profile because it showed the clearest pattern at different time points. 55.0 genes belong to this profile and 22.0 genes were expected to belong to profile 48. The p-value for the enrichment of genes in this profile is 2.2 E-9.

← Older revision		Revision as of 20:40, 3 November 2017
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−	==Powerpoint==	+	==== Clustering and GO Term Enrichment with stem ====
		+
		+	# '''Prepare your microarray data file for loading into STEM.'''
		+	#* Download your Excel workbook that you used for your [[Week 8]] assignment.
		+	#* Insert a new worksheet into your Excel workbook, and name it "(STRAIN)_stem".
		+	#* Select all of the data from your "dGLN3_ANOVA" worksheet and Paste special > paste values into your "dGLN3_stem" worksheet.
		+	#** Your leftmost column should have the column header "Master_Index". Rename this column to "SPOT". Column B should be named "ID". Rename this column to "Gene Symbol". Delete the column named "Standard_Name".
		+	#** Filter the data on the B-H corrected p value to be > 0.05 (that's '''greater than''' in this case).
		+	#*** Once the data has been filtered, select all of the rows (except for your header row) and delete the rows by right-clicking and choosing "Delete Row" from the context menu. Undo the filter. This ensures that we will cluster only the genes with a "significant" change in expression and not the noise. '''''Record the number of genes left in your electronic notebook.'''''
		+	#** Delete all of the data columns '''''EXCEPT''''' for the Average Log Fold change columns for each timepoint (for example, wt_AvgLogFC_t15, etc.).
		+	#** Rename the data columns with just the time and units (for example, 15m, 30m, etc.).
		+	#** Save your work. Then use ''Save As'' to save this spreadsheet as Text (Tab-delimited) (*.txt). Click OK to the warnings and close your file.
		+	#*** Note that you should turn on the file extensions if you have not already done so.
		+	# '''Now download and extract the STEM software.''' [http://www.cs.cmu.edu/~jernst/stem/ Click here to go to the STEM web site].
		+	#* Click on the [http://www.andrew.cmu.edu/user/zivbj/stemreg.html download link], register, and download the <code>stem.zip</code> file to your Desktop.
		+	#* Unzip the file. In Seaver 120, you can right click on the file icon and select the menu item ''7-zip > Extract Here''.
		+	#* This will create a folder called <code>stem</code>. Inside the folder, double-click on the <code>stem.jar</code> to launch the STEM program.
		+	<!--#** In Seaver 120, we encountered an issue where the program would not launch on the Windows XP machines due to a lack of memory. (Even though the computers have been upgraded to Windows 7, do this to launch the program.) To get around this problem, launch STEM from the command line.
		+	#*** Go to the start menu and click on ''Programs > Accessories > Command Prompt''.
		+	#*** You will need to navigate to the directory (folder) in which the STEM program resides. If you followed the instructions above and extracted the stem folder to the Desktop, type the following: <code>cd Desktop\stem</code> and press "Enter".
		+	#*** To launch the program then type: <code>java -mx512M -jar stem.jar -d defaults.txt</code> and press "Enter". This will launch the program with less memory allocated to it.-->
		+	# '''Running STEM'''
		+	## In section 1 (Expression Data Info) of the the main STEM interface window, click on the ''Browse...'' button to navigate to and select your file.
		+	##* Click on the radio button ''No normalization/add 0''.
		+	##* Check the box next to ''Spot IDs included in the data file''.
		+	## In section 2 (Gene Info) of the main STEM interface window, select ''Saccharomyces cerevisiae (SGD)'', from the drop-down menu for Gene Annotation Source. Select ''No cross references'', from the Cross Reference Source drop-down menu. Select ''No Gene Locations'' from the Gene Location Source drop-down menu.
		+	## In section 3 (Options) of the main STEM interface window, make sure that the Clustering Method says "STEM Clustering Method" and do not change the defaults for Maximum Number of Model Profiles or Maximum Unit Change in Model Profiles between Time Points.
		+	## In section 4 (Execute) click on the yellow Execute button to run STEM.
		+	# '''Viewing and Saving STEM Results'''
		+	## A new window will open called "All STEM Profiles (1)". Each box corresponds to a model expression profile. Colored profiles have a statistically significant number of genes assigned; they are arranged in order from most to least significant p value. Profiles with the same color belong to the same cluster of profiles. The number in each box is simply an ID number for the profile.
		+	##* Click on the button that says "Interface Options...". At the bottom of the Interface Options window that appears below where it says "X-axis scale should be:", click on the radio button that says "Based on real time". Then close the Interface Options window.
		+	##*Take a screenshot of this window (on a PC, simultaneously press the <code>Alt</code> and <code>PrintScreen</code> buttons to save the view in the active window to the clipboard) and paste it into a PowerPoint presentation to save your figures.
		+	## Click on each of the SIGNIFICANT profiles (the colored ones) to open a window showing a more detailed plot containing all of the genes in that profile.
		+	##* Take a screenshot of each of the individual profile windows and save the images in your PowerPoint presentation.
		+	##* At the bottom of each profile window, there are two yellow buttons "Profile Gene Table" and "Profile GO Table". For each of the profiles, click on the "Profile Gene Table" button to see the list of genes belonging to the profile. In the window that appears, click on the "Save Table" button and save the file to your desktop. Make your filename descriptive of the contents, e.g. "wt_profile#_genelist.txt", where you replace the number symbol with the actual profile number.
		+	##** Upload these files to the wiki and link to them on your individual journal page. (Note that it will be easier to zip all the files together and upload them as one file).
		+	##* For each of the significant profiles, click on the "Profile GO Table" to see the list of Gene Ontology terms belonging to the profile. In the window that appears, click on the "Save Table" button and save the file to your desktop. Make your filename descriptive of the contents, e.g. "wt_profile#_GOlist.txt", where you use "wt", "dGLN3", etc. to indicate the dataset and where you replace the number symbol with the actual profile number. At this point you have saved all of the primary data from the STEM software and it's time to interpret the results!
		+	##** Upload these files to the wiki and link to them on your individual journal page. (Note that it will be easier to zip all the files together and upload them as one file).
		+	# '''Analyzing and Interpreting STEM Results'''
		+	## Select '''''one''''' of the profiles you saved in the previous step for further intepretation of the data. I suggest that you choose one that has a pattern of up- or down-regulated genes at the cold shock timepoints. '''''Each member of your group should choose a different profile.''''' Answer the following:
		+	##* '''''Why did you select this profile? In other words, why was it interesting to you?'''''
		+	##* '''''How many genes belong to this profile?'''''
		+	##* '''''How many genes were expected to belong to this profile?'''''
		+	##* '''''What is the p value for the enrichment of genes in this profile?''''' Bear in mind that we just finished computing p values to determine whether each individual gene had a significant change in gene expression at each time point. This p value determines whether the number of genes that show this particular expression profile across the time points is significantly more than expected.
		+	##* Open the GO list file you saved for this profile in Excel. This list shows all of the Gene Ontology terms that are associated with genes that fit this profile. Select the third row and then choose from the menu Data > Filter > Autofilter. Filter on the "p-value" column to show only GO terms that have a p value of < 0.05. '''''How many GO terms are associated with this profile at p < 0.05?''''' The GO list also has a column called "Corrected p-value". This correction is needed because the software has performed thousands of significance tests. Filter on the "Corrected p-value" column to show only GO terms that have a corrected p value of < 0.05. '''''How many GO terms are associated with this profile with a corrected p value < 0.05?'''''
		+	##* Select 6 Gene Ontology terms from your filtered list (either p < 0.05 or corrected p < 0.05).
		+	##** Each member of the group will be reporting on his or her own cluster in your presentation next week. You should take care to choose terms that are the most significant, but that are also not too redundant. For example, "RNA metabolism" and "RNA biosynthesis" are redundant with each other because they mean almost the same thing.
		+	##**'''''Look up the definitions for each of the terms at [http://geneontology.org http://geneontology.org]. In your final presentation, you will discuss the biological interpretation of these GO terms. In other words, why does the cell react to cold shock by changing the expression of genes associated with these GO terms? Also, what does this have to do with the transcription factor that was deleted from your strain?'''''
		+	##** To easily look up the definitions, go to [http://geneontology.org http://geneontology.org].
		+	##** Copy and paste the GO ID (e.g. GO:0044848) into the search field at center top of the page called "Search GO Data".
		+	##** In the [http://amigo.geneontology.org/amigo/medial_search?q=GO%3A0044848 results] page, click on the button that says "Link to detailed information about <term>, in this case "biological phase"".
		+	##** The definition will be on the next results page, e.g. [http://amigo.geneontology.org/amigo/term/GO:0044848 here].
		+
		+
		+	==Powerpoint and Zip==
	[[Media:StemGraphResults.pptx\|dGLN3 Stem Graph Result Powerpoint]]		[[Media:StemGraphResults.pptx\|dGLN3 Stem Graph Result Powerpoint]]
		+	[[Media:DGLN3_STEM.zip\|DGLN3 Zip File]]